Microfabrication of biomedical lab-on-chip devices. A review

Estonian Journal <strong>of</strong> Engineering, 2011, 17, 2, 109–139 doi: 10.3176/eng.2011.2.03
<strong>Micr<strong>of</strong>abrication</strong> <strong>of</strong> <strong>biomedical</strong> <strong>lab</strong>-on-chip devices.
A review
Athanasios T. Giannitsis
Department <strong>of</strong> Electronics, Tallinn University <strong>of</strong> Technology, Ehitajate tee 5, 19086 Tallinn,
Estonia; thanasis@elin.ttu.ee
Received 1 February 2011, in revised form 8 April 2011
Abstract. Lab-on-chip systems are a class <strong>of</strong> miniaturized analytical devices that integrate fluidics,
electronics and various sensorics. They are capable <strong>of</strong> analysing biochemical liquid samples, like
solutions <strong>of</strong> metabolites, macromolecules, proteins, nucleic acids, or cells and viruses. Supplementary
to their measuring capabilities, the <strong>lab</strong>-on-chip devices facilitate fluidic transportation,
sorting, mixing, or separation <strong>of</strong> liquid samples. A type <strong>of</strong> <strong>lab</strong>-on-chip devices, named biochip, is
devoted specifically to genomic, proteomic and pharmaceutical tests. The significance <strong>of</strong> such
miniaturized devices lies in their potentiality <strong>of</strong> automating <strong>lab</strong>oratory procedures, which highly
reduces the time <strong>of</strong> <strong>biomedical</strong> tests and <strong>lab</strong>oratory work. This review summarizes numerous
fabrication methods and procedures for producing <strong>lab</strong>-on-chip devices and also envisages future
evolution.
Key words: <strong>lab</strong>-on-chip devices, biosensors, biochips, micr<strong>of</strong>luidics, micr<strong>of</strong>abrication.
1. INTRODUCTION
Lab-on-chip technology focuses on the development <strong>of</strong> hybrid devices, which
integrate fluidic and electronic components onto the same chip [ 1–5 ]. They are
devoted primarily to liquid sample testing and handling. Such devices reduce
<strong>lab</strong>oratory processes in a manner competitive to bench-top instruments. Lab-onchip
technology emphasizes integration, chip programmability, increased
sensitivity, minimal reagent consumption, sterilization and efficient sample
detection and separation. Lab-on-chip functionality includes sample handling,
mixing, filtering, analysis and monitoring. A typical <strong>lab</strong>-on-chip device contains
microchannels, which allow liquid samples to flow inside the chip, but also
integrates measuring, sensing and actuating components such as microvalves,
micr<strong>of</strong>luidic mixers, microelectrodes, thermal elements, or optical apparatuses.
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Some commercial <strong>lab</strong>-on-chip devices incorporate electronics that operate their
apparatuses (Fig. 1). The <strong>lab</strong>-on-chip devices are considered analogous to the
systems-on-chip in electronics.
Biochemists, synthetic chemists and medical doctors currently evaluate the
potentiality <strong>of</strong> <strong>lab</strong>-on-chip devices in the context <strong>of</strong> biochemical synthesis,
analysis and screening. Specific areas <strong>of</strong> applications have emerged, from the
detection <strong>of</strong> infectious diseases and diagnostics to the assessment <strong>of</strong> food quality.
Several on-chip clinical assessments include cell analysis, cytometry, blood
analysis, nucleic acids amplification, genetic mapping, enzymatic assays, peptide
analysis, protein separation, toxicity analysis and bioassays. Lab-on-chip devices
find growing applications in drug discovery. In pharmacy, the <strong>lab</strong>-on-chip
devices gradually become valuable in the area <strong>of</strong> drug research with the emphasis
on cell targeting, clinical trials, drug synthesis, pharmaceutical formulations and
product management process. The <strong>lab</strong>-on-chip devices are found promising in the
analysis <strong>of</strong> drugs and determination <strong>of</strong> optimal dosages. This is especially useful
for testing the synergistic effect <strong>of</strong> combined drugs. Micr<strong>of</strong>luidic networks-onchip
<strong>of</strong>fer unique opportunities to imitate natural veins for testing nanoparticles
as drug carriers for targeting cells or, moreover, they <strong>of</strong>fer opportunities for
examining in vitro the metabolisms <strong>of</strong> biological cultures. Technical limitations
such as size reduction, sample input rates, power consumption, but also chip
reliability and biocompatibility shall all be accounted in the design <strong>of</strong> <strong>lab</strong>-on-chip
devices.
Lab-on-chip devices have become very attractive nowadays as they force the
development <strong>of</strong> personalized devices for point-<strong>of</strong>-care treatments [ 6 ]. The <strong>lab</strong>-onchip
technology enables the development <strong>of</strong> the next generation <strong>of</strong> portable and
implantable bioelectronic devices. Due to their biosensing capability and embedding
concept, the <strong>lab</strong>-on-chip systems are attractive platforms for developing
implantable bio-inspired sensors.
Fig. 1. Essential dataflow functionality <strong>of</strong> <strong>lab</strong>-on-chip devices. It can be realized with electronics,
incorporated either in the micr<strong>of</strong>luidic chip or on separate circuit boards, attachable to the chip. The
sensor is followed by an analogue front-end, which conditions the measuring signal, analogue-todigital
converters (ADC), and a digital signal processor that analyses the signal. Depending on the
measuring method the signals can be electrical, optical, etc. The analysed data can be further sent
via a bus to external computer for post-processing, or even visualized on integrated displays or
external screen. Additionally to processing measurement data, the processor may adjust the state <strong>of</strong>
the measurand sample via mechanical, electrical, or optical actuation.
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Lab-on-chip technology has adopted most analytical chemistry methods, such
as electrochemical sensing [ 7,8 ], mass spectrometry [ 9,10 ], thermal detection, ultrasound
waves [ 11 ], capillary electrophoresis [ 12–17 ], electrochromatography [ 18–24 ],
and moreover optical methods like absorbance [ 25,26 ], infrared and Raman spectroscopy
[ 27 ], scattering [ 28 ], refractive index [ 29 ], surface plasmon resonance [ 30 ],
plasma emission and fluorescence [ 31 ].
2. ADVANTAGES OF LAB-ON-CHIP DEVICES
Built to automate <strong>lab</strong>oratory processes, some notable technical advantages <strong>of</strong>
<strong>lab</strong>-on-chip devices are compactness, portability, modularity, reconfigurability,
embedded computing, automated sample handling, low electronic noise, limited
power consumption and straightforward integration <strong>of</strong> their components. In
addition to minimal quantity request for samples, analytes, or reagents, the <strong>lab</strong>on-chip
devices are fully enclosed and this reduces contamination <strong>of</strong> the carried
samples. Furthermore, <strong>lab</strong>-on-chip devices are capable <strong>of</strong> supporting a wide
range <strong>of</strong> processes such as sampling, routing, transport, dispensing and mixing,
mostly with reduced moving or spinning components, therefore the usability and
lifespan <strong>of</strong> the device is increased. Due to their small size, the <strong>lab</strong>-on-chip
devices <strong>of</strong>fer precise fluidic transportation via the use <strong>of</strong> electrokinetics or micropumping,
efficient separation <strong>of</strong> the liquid samples and precision in the measurement
<strong>of</strong> samples.
Although usually the fluidic transportation pertains continuous flows, dropletbased
segmented flows are also favoured [ 32–34 ]. The droplets usually find
applications as small bioreactors for cellular and molecular assays for advanced
therapeutics [ 35–37 ]. Supplementary, and depending on the particularity <strong>of</strong> the
application, tiny droplets can be employed as electrical [ 38 ], optical [ 39,40 ] or
thermal agents. The benefit for biomedicine is the possibility for continuous and
automated monitoring <strong>of</strong> biochemical samples and the possibility <strong>of</strong> adjusting
procedures by online feedbacks. Due to the small fluidic volumes that they
handle, the <strong>lab</strong>-on-chip devices can reduce the time <strong>of</strong> synthesis <strong>of</strong> a product and
the time <strong>of</strong> analysis <strong>of</strong> a sample. They can measure samples with greater precision,
but most essential is their capability <strong>of</strong> controlling the chemical reactions
through efficient control <strong>of</strong> the reactants concentration. The <strong>lab</strong>-on-chip devices
can either provide cascade or parallel sample processing. The advantage <strong>of</strong>
parallel processing allows simultaneous tests <strong>of</strong> different samples with different
reagents, so that the product’s effectiveness can be characterized efficiently.
Lab-on-chip technology <strong>of</strong>fers affordable and simple maintenance <strong>of</strong> the
fluidic chips. They can be easily cleaned and sterilized with cleaning solutions
like sodium hydroxide, nitric acid, decanol, ethanol, bleach and ethylene
oxide [ 1 ]. Alternatively, ultraviolet radiation, autoclaving and heat can sterilize
the devices. Furthermore, capillary plasma can remove organic remains from
inside the fluidic chips.
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Key manufacturing advantages that make <strong>lab</strong>-on-chip devices affordable are:
achievable mass production, affordable replacement cost, short time manufacture,
simple quality tests, and broad range <strong>of</strong> supporting computer aided
design and simulation s<strong>of</strong>tware tools. Most <strong>of</strong> the advantages <strong>of</strong> the fluidic <strong>lab</strong>on-chip
devices are analogous to those <strong>of</strong> the integrated electronic chips.
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3. APPLICATIONS OF LAB-ON-CHIP DEVICES
Clinical medicine greatly benefits from <strong>lab</strong>-on-chip technology as it suites for
drug tests, tests for observing pandemics, glucose monitoring, diabetic control,
diagnosis <strong>of</strong> diseases and numerous other tests. Lab-on-chip devices enhance
numerous <strong>biomedical</strong> tests that entail mixing, analysis and separation <strong>of</strong> samples,
which usually consist <strong>of</strong> cell suspensions, nucleic acids, proteins, etc. Analytical,
electrical, or optical detection methods are possible. The electrical detection
methods depend exclusively on the polar properties <strong>of</strong> the molecules <strong>of</strong> the liquid
samples. For example, carbon dioxide levels, oxygen levels, or pH values can be
measured electrochemically. On the contrary, most analytical or optical
techniques require <strong>lab</strong>elling, which entails chemoluminescence, fluorescence, or
radioactive markers. Most separation methods <strong>of</strong> <strong>lab</strong>-on-chip systems are
miniaturized approaches <strong>of</strong> larger ones. There are diverse screening methods,
which <strong>of</strong>fer high sample throughput, whereas other methods <strong>of</strong>fer reliability and
precision. The separation <strong>of</strong> biomolecules, cells, or nanoparticles can be managed
by transportation methods, which are based on the charge and size <strong>of</strong> the
substances. These transportation methods can be the following ones:
(a) hydrodynamic manipulation, which employs hydrodynamic pressure [ 41–44 ];
(b) electrical manipulation which transports electrolytes, suspensions <strong>of</strong> particles
or cells, or entire aqueous volumes like droplets, via the use <strong>of</strong> electrokinetic
mechanisms such as electrophoresis [ 45 ], dielectrophoresis [ 46,47 ] and electrowetting
[ 48–51 ];
(c) magnetophoresis that employs magnetic fields in conjunction with magnetic
nanoparticles suspended in the samples [ 52 ];
(d) optical manipulation that employs laser-light pressure which moves nanoparticles
or tinny droplets [ 29,33 ]; alternatively, light actuation can alter locally
the degree <strong>of</strong> hydrophobicity <strong>of</strong> a surface and consequently direct aqueous
volumes via hydrophilic routes.
Electrical and mechanical micropumps are largely employed in micr<strong>of</strong>luidic
manipulation [ 53 ]. Electric micropumps utilize electrokinetics [ 54 ], piezoelectrics
[ 55,56 ], or magnetohydrodynamics [ 57 ], while mechanical micropumps
utilize hydrodynamic pressure [ 58 ], thermal expansion [ 59 ], osmotic pressure [ 60 ],
or other transducer or induced forces. Electrocapillary, an important electrokinetic
pumping mechanism, boosted the development <strong>of</strong> <strong>lab</strong>-on-chip devices
[ 13,61,62 ]. This achievement caused the realization <strong>of</strong> miniaturized analytical
instruments, namely on-chip chromatographic systems [ 63 ]. Other pumping

mechanisms employ thermal gradients, or magnetophoresis. Fluidic <strong>lab</strong>-on-chip
devices can facilitate cell screening [ 64–66 ]. This works on the basis <strong>of</strong> forcing
cells into specific streamlines, separates them by size, and drives them into
specific outlets [ 67 ]. This finds applications in blood cell separation, where
erythrocytes flow right in the centre <strong>of</strong> a microcapillary and the leukocytes, due
to their differing mass, pursue streamlines along the capillary wall [ 43,68 ]. Besides
hydrodynamic separation, microvalves can sort suspensions <strong>of</strong> cells or nanoparticles,
usually in conjunction with electrokinetic actuation methods [ 69–71 ].
Electrically actuated microvalves are made <strong>of</strong> electroactive elastomers or piezoelectrics,
which can be embedded easily in fluidic chips. Piezoelectric micropumps
are activated when voltage applies on a piezoelectric material causing
expansion. The expansion can be several micrometres, but in the microscale this
causes significant pressure, which pushes the fluid adequately. Magnetically
actuated microvalves also exist, made <strong>of</strong> magnetically inductive materials. The
magnetic actuation field can be produced on-chip by means <strong>of</strong> current patterns,
integrated on the chip. Lab-on-chip devices are efficient in mixing samples in a
control<strong>lab</strong>le and precise manner due to the use <strong>of</strong> tiny liquid volumes. Examples
<strong>of</strong> mixers include distributive mixers, static mixers, T-junction mixers and vortex
mixers. Active mixers consume power in order to increase the interfacial area
between the mixing fluids. Examples <strong>of</strong> active mixers include electrokinetic
mixers, chaotic advection mixers and magnetically driven mixers. Passive mixing
can be achieved in microchannels with structures called twists that cause
turbulence. Some mixers are more efficient at faster flow rates, whereas others
work more efficiently at slower flow rates [ 32 ].
Temperature control is important in micr<strong>of</strong>luidic devices. Temperature
gradients can be generated inside micr<strong>of</strong>luidic channels by means <strong>of</strong> miniaturized
heating elements, such as heat exchangers, heaters or coolers [ 72 ]. The efficiency
<strong>of</strong> micro-heat exchangers depends on their ability to regulate the temperature <strong>of</strong>
the transported fluid, in conjunction with reservoirs that buffer rapid changes <strong>of</strong>
temperature. The micro-heaters are assembled in the form <strong>of</strong> coils. Due to the
inherently small dimensions <strong>of</strong> the micr<strong>of</strong>luidic devices, the thermal coupling
between a micro-heater and a reservoir is very efficient. Coolers can be made out
<strong>of</strong> heatsinks that induct the heat to the environment. The combination <strong>of</strong> small
fluidic volumes and the precision <strong>of</strong> the heatsinks allow cooling to ambient
temperature with ease. Repeatable temperature cycles are essential in the process
for nucleic acid amplification and find application in the polymerase chain
reaction (PCR) arrays-on-chips [ 72–77 ]. PCR replicates small amounts <strong>of</strong> nucleic
acids into large amounts. PCR involves a three-step thermal cycle that promotes
the replication: heating (90–95 °C), cooling (50 °C), and slight warming (60–
70 °C). PCR chips include various temperature zones whose benefit is that the
cycle time no longer relies on the time required to heat or cool the sample. The
benefit <strong>of</strong> performing PCR in micr<strong>of</strong>luidic chips is due to the very small liquid
volumes used, for which temperature homogeneity and diffusion efficiency are
higher in comparison with ordinary <strong>lab</strong>oratory vessels. However, thermal cycles
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may induce undesirable effects, especially regarding evaporation and bubble
formation. The droplet-based biochips eliminate most <strong>of</strong> these defects during
PCR processes due to the isolation <strong>of</strong> the droplets by a second phase oleic
fluid [ 78,79 ]. Fully automated PCR biochips that contain mixers, heaters, and
microarray sensors are capable <strong>of</strong> performing sequential functions like hybridization,
preconcentration, purification, lysis, and electrochemical assessment <strong>of</strong>
complex biological solutions [ 80 ]. In bio-analysis, biosensors, integrated in micr<strong>of</strong>luidic
devices, <strong>of</strong>fer possibilities for electrochemical or optical analysis <strong>of</strong>
samples. Enzyme-based electrochemical biosensors can measure oxygen consumption
or pH production by means <strong>of</strong> enzymatic reactions. The enzymes
catalyse the reactions <strong>of</strong> the analytes and the sensor measures the products [ 81 ]. In
the field <strong>of</strong> bioanalytical separation, the capillary chromatographic chips adopt
fluorescent polystyrene nanoparticles or <strong>lab</strong>elled macromolecules to enhance cell
or protein separation by image recognition [ 18,82,83 ]. In analogy, the ion exchange
chromatographic chips separate electrokinetically analytes such as polar
molecules, proteins, nucleotides and amino acids according to their ionic charge.
Genomic research requires analysis, which can be utilized by means <strong>of</strong>
specialized fluidic chips called microarrays, which are arrangements <strong>of</strong> open
planar arrays that each contains thousands <strong>of</strong> specific oligonucleotides, covalently
bonded on glass or silicon substrate [ 84–87 ]. These oligonucleotides function as
probes, which hybridize the DNA fragments that contact the microarray, by
forming hydrogen bonds between the DNA fragments and the complementary
nucleotide base pairs <strong>of</strong> each probe. A microarray can accomplish many
genotype tests in parallel. The detection method in microarray chips is based on
microscopy analysis via the use <strong>of</strong> fluorophore or chemoluminescence <strong>lab</strong>els that
determine abundance <strong>of</strong> nucleic acid sequences. The <strong>lab</strong>-on-chip microarrays can
be distinguished between cDNA-microarrays and oligonucleotide-microarrays.
The latter can perform genotyping and resequencing [ 88 ]. The microarray chips
can be employed in nucleic acid analysis, which includes DNA extraction and
purification, amplification, hybridization, sequencing, gene expression analysis,
genotyping and DNA separation. Moreover, microarrays can recombine
nucleotide fragments by employing endonucleases to cleave and rebind the fragments
with DNA ligase enzymes. Fluorescence microscopy is mainly employed
in microarrays because <strong>of</strong> its challenging limits <strong>of</strong> detection, due to its sensitivity,
which approaches concentrations <strong>of</strong> picomoles. Detection sensitivity <strong>of</strong> picomoles
is appropriate in most applications that require high sensitivity, such as
DNA sequencing and many immunoassays [ 89 ]. There are great potential applications
<strong>of</strong> microarray chips in biotechnology as well as <strong>of</strong> DNA-inspired <strong>lab</strong>-onchips
in nanotechnology [ 90 ]. The oxidative type <strong>of</strong> DNA-mediated charge transport
has significant results in mutagenesis, apoptosis, or cancer studies. On the
other hand, excess electron transport plays a growing role in the development <strong>of</strong>
electrochemical DNA chips for the detection <strong>of</strong> DNA base mutations [ 91 ].
Knowledge about excess electron transport in DNA has potentials for nanotechnological
applications, such as supramolecular electronics. In this field, the
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esearch focuses on the role <strong>of</strong> DNA as supramolecule that features important
properties for nanowires [ 92 ].
Lab-on-chip devices support cell detection by means <strong>of</strong> capillary electrophoresis,
electroporation, cytometry or electrical impedance. The cells can be
cultivated within aqueous microreactors inside the chips [ 35,69,70,93 ]. With <strong>lab</strong>-onchip
devices one can measure accurately chemical stimuli on cells, as cellular
signals are weak and not easily detected with conventional analytical methods.
Cytometers allows cell counting and analysis <strong>of</strong> cellular parameters like size
distribution or growth rate. Cytometry commonly employs electrical impedance
methods [ 94–96 ], but also employs fluorescence microscopy; however, fluorescence
microscopy requires cell <strong>lab</strong>elling with dyes or fluorescent nanoparticles
and external optical apparatuses, such as optical fibres and illuminators [ 97 ].
Other electrokinetic methods rely on contactless conductivity detection <strong>of</strong>
organic ions or electrolytes where typically two on-chip embedded tubular
electrodes, away from each other, measure changes <strong>of</strong> the ohmic resistance <strong>of</strong> the
solution that flows inside the micr<strong>of</strong>luidic channel [ 8,15,17,98–101 ]. Capillary electrophoresis
performs electroseparation <strong>of</strong> the ionic compounds, based on the size-tocharge
ratio <strong>of</strong> the ions, which are transported electrokinetically in the carrier
liquid. The electrocapillary forces drag the ions into the capillary channel while
the fluidic sample approaches the inlet <strong>of</strong> the microchannel [ 102–104 ]. Capillary
electrophoresis is capable <strong>of</strong> separating particulate substances like cells, amino
acids, proteins, peptides, mitochondria, or bacteria [ 105 ]. In micr<strong>of</strong>luidic channels
the surface-to-volume increases significantly due to reduced dimensions, and
electrophoresis force becomes more efficient due to the shortening <strong>of</strong> the
distance. In capillary electrophoresis devices the electric fields range to some
hundreds V/cm with pulse durations <strong>of</strong> milliseconds. Capillary electrophoresis
separation is fast and robust and high throughput can be obtained. Electroporation
is an electrical method that accesses the interior <strong>of</strong> cells. Applying short
and intense electric pulses increases the conductivity and transiently permeabilizes
the lipid membrane <strong>of</strong> a cell and introduces substances into the cytoplasm.
Electroporation is used for inserting drugs or genes into cells and is highly
efficient when performed in vitro in <strong>lab</strong>-on-chip devices [ 106 ]. Electrokinetic
manipulation <strong>of</strong> suspended particles by means <strong>of</strong> electric fields is widely used in
<strong>lab</strong>-on-chip devices for in vitro separation <strong>of</strong> particles or cells [ 107,108 ]. Dielectrophoresis
manipulates and separates cells, beads or nanoparticles by means <strong>of</strong>
inhomogeneous electric fields that can be produced with electrodes <strong>of</strong> specific
shapes [ 46,47 ]. The cells can be collected or directed away from the electrodes
under the influence <strong>of</strong> the dielectrophoresis force, which is directed towards the
field gradient, and originates from the permittivity difference between the carrier
fluid and cells [ 109 ]. The possibility <strong>of</strong> using dielectrophoresis for in vitro separation
<strong>of</strong> blood cells, based on their conductivity characteristics, can automate
partition <strong>of</strong> white blood cells from erythrocytes. Several human cancer cells have
been successfully studied and dielectrophoretically sorted [ 110–113 ]. Dielectrophoresis
can further measure dielectric differences <strong>of</strong> cells or particles by means
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<strong>of</strong> electrorotational spectra [ 114 ]. Cancerous cells are different from healthy
erythrocytes and lymphocytes. Their dielectric diversification can be exploited
for removing metastatic cancer cells from healthy blood cells. Other electrokinetic
means with growing applications in <strong>lab</strong>-on-chips technology is electroosmotic
flow, a coulomb effect that results in transportation <strong>of</strong> ions <strong>of</strong> an electric
double layer [ 115,116 ], and its reverse effect, streaming potential, that is induced
voltage on an electrode due to the flowing motion <strong>of</strong> counterions <strong>of</strong> an electric
double layer [ 117,118 ].
Today, magnetic separation devices are avai<strong>lab</strong>le and magnetic cell separation
<strong>of</strong>fers diagnostic and therapeutic values [ 119 ]. Magnetophoretic sorters can
capture cells that are bonded with magnetic beads, or nanoparticle aggregates,
and force them flow into the carrier fluid with increased selectivity [ 52,120 ]. Moreover,
magnetic nanoparticles can be used to destruct targeted cells by penetration,
or to release carried drug into the cell’s cytoplasm [ 121 ]. The magnetic field can
be generated by current patterns on the chip. An important advantage <strong>of</strong> magnetic
actuators in comparison with the electric ones is due to the magnitude <strong>of</strong> the
magnetic driving force that is significantly larger in comparison to electrokinetic
forces. This highly improves the control <strong>of</strong> the liquid flow. The possibility <strong>of</strong>
incorporating magnetic nanoparticles into cells as inert tracers for cell monitoring,
allows measuring cytoskeleton associated cell functions [ 122,123 ]. Within
cytoplasm the magnetic nanoparticles may equilibrate, but intracellular transports
disorient the magnetic nanoparticles resulting in the decay <strong>of</strong> magnetization
which can be recorded by magneto-impedimetric sensor, embedded in the chip.
Magnetic actuators can transport aqueous droplets that enclose magnetic nanoparticles
[ 124 ]. Under the influence <strong>of</strong> the magnetic field the magnetic nanoparticles
can drag the droplet and execute all the usual micr<strong>of</strong>luidic operations
such as displacement, merging, mixing and separation [ 125 ].
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4. DESIGNING AND MANUFACTURING PROCESS
The manufacturing process for producing a <strong>lab</strong>-on-chip device is described in
Fig. 2. A numerical simulation that models the performance <strong>of</strong> a <strong>lab</strong>-on-chip
device is essential before manufacturing the device. Simulations are important,
Fig. 2. Flow diagram <strong>of</strong> the manufacturing steps required for producing a commercial <strong>lab</strong>-on-chip
device.

ecause they imitate the functionality <strong>of</strong> the components <strong>of</strong> the device and then
use the simulation data to infer the real-world functionality and performance.
Numerical simulations usually rely on finite element algorithms that can be
actualized by specialized finite element s<strong>of</strong>tware. Fully dynamic simulations are
computationally not efficient, because they require huge computational
resources. Alternatively, the numerical simulations can be performed quasistatically,
but this does not provide information about the dynamic behaviour <strong>of</strong> a
component. In order to study the dynamics <strong>of</strong> a component in a computationally
efficient way, it is essential to reduce the number <strong>of</strong> degrees <strong>of</strong> freedom, from a
three-dimensional model to a two-dimensional model. Due to the hierarchical
structure <strong>of</strong> <strong>lab</strong>-on-chip devices, a numerical simulation requires studies <strong>of</strong>
sca<strong>lab</strong>ility [ 2 ]. A <strong>lab</strong>-on-chip maker must handle heterogeneous and multiple
components and should deal with complex fluidic flows and multiple component
simulation. It is important for the chipmaker to investigate how the performance
<strong>of</strong> a micr<strong>of</strong>luidic device scales with increasingly complicated sample analysis. In
addition, it is necessary to investigate how the performance <strong>of</strong> the <strong>lab</strong>-on-chip
system balances with advances in the present technology. Although a number <strong>of</strong>
computer-aided design tools, dedicated for <strong>lab</strong>-on-chip designs, exist, any usual
computer-aided design tool that supports multilayer drawing and vector graphics
can be used.
Lab-on-chips devices should be modular to allow, to the most possible extent,
expansion to varied functions. The goal is to leverage system-level and component-level
technology, including fluidics and sensorics, to be expanded, reconfigured
and reused for varieties <strong>of</strong> applications on the same chip. Usually data
analysis s<strong>of</strong>tware can be reconfigured. For example, a <strong>lab</strong>-on-chip microarray
device should be capable <strong>of</strong> processing genotyping and gene expression applications.
A chromatographic fluidic biosensor or a temperature-based actuator
should be capable <strong>of</strong> processing liquids and gases. An electrokinetic actuator
should be capable <strong>of</strong> processing both continuous or segmented fluidic samples
and the same device be competent <strong>of</strong> separating nanometer-size macromolecules
by means <strong>of</strong> electrophoresis, or separating micrometer-size cells by means <strong>of</strong>
dielectrophoresis, or separating millimetre-scaled droplets that enclose cells, by
means <strong>of</strong> electrowetting. Segmented flows that consist <strong>of</strong> droplet microreactors
allow dynamic reconfigurability, because the cells, cultured inside each droplet,
can be reconfigured to different functionality within each individual droplet.
The system architecture <strong>of</strong> a micr<strong>of</strong>luidic chip should assort the elementary
fluidic components like micropumps, microvalves, reservoirs, microchannels,
nozzles and junctions in a manner that allows a functional and straightforward
process. It is convenient to consider circuit analogies when analysing micr<strong>of</strong>luidic
<strong>lab</strong>-on-chips: the reservoirs are comparable to registers as they are storage
components. Microchannels are equivalent to wires as they carry flows. Microvalves
are analogous to gates as they provide selectivity. Micropumps are
analogous to voltage sources as they actuate the flow.
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Electromagnetic compatibility issues should be carefully considered in <strong>lab</strong>on-chip
design because measuring and control signals usually interfere. To
eliminate electromagnetic interferences, usually timeslot sharing is required.
Furthermore, the metal holder <strong>of</strong> a <strong>lab</strong>-on-chip should be designed in a way that
shields the device. The electronics and embedded circuits that integrate onto a
<strong>lab</strong>-on-chip device should comply with the electromagnetic compatibility
standards <strong>of</strong> radio frequency electronics. Any circuit board, attached on the chip
holder, should be designed in a way that minimizes electrical connections. This is
particularly important in the impedimetric <strong>lab</strong>-on-chip devices, which operate at
radio frequencies, where the electric length <strong>of</strong> the connections influences the
measurement <strong>of</strong> the impedance values.
The <strong>lab</strong>-on-chip devices should be designed in a manner to be easily
maintained. A device should <strong>of</strong>fer easy channel washing and sample filling. The
chip holders and the other connecting components should be easily handled,
assembled and used by the <strong>lab</strong>oratory staff. Many <strong>lab</strong>-on-chip devices might be
requested to fulfil specific customized tasks, but still the manufacturer should
design the device in a manner that provides the user the fundamentals for handy
maintenance, calibration and operation.
The manufacturing procedure for producing <strong>lab</strong>-on-chip devices is comparable
to the microelectronic chip fabrication procedure. It involves photolithography,
where patterns that represent micr<strong>of</strong>luidic and electric features are
transferred onto a photosensitive substrate by means <strong>of</strong> ultraviolet illumination
via a chromium photomask. Then chemical etching reveals the elements.
Application <strong>of</strong> photolithography via successive photomasks produces multilayered
structures. Photolithography has continuous improvements in the ability
<strong>of</strong> resolving ever-smaller features, but also in producing high-aspect-ratio
features. This is especially important in the fabrication <strong>of</strong> micr<strong>of</strong>luidic channels.
Each implementation <strong>of</strong> the photolithographic process has its own specific
requirements, but there is common sequence, which involves the following, as
shown in Fig. 3.
(a) Metallization <strong>of</strong> the substrate by sputtering gold or platinum film on it.
Electrodes and other conductive elements can be shaped on this metallized
surface.
(b) Spin-coating <strong>of</strong> photoresist onto the metallized surface, at typical speeds
(depending on the resist’s viscosity) <strong>of</strong> 1500–8000 rpm, and heat at 100 °C to
dry the resist. The photoresist serves as an intermediate layer via which the
patterns can be transferred onto the metallized substrate.
(c) Exposure <strong>of</strong> the photoresist layer to ultraviolet light that results in the
transfer <strong>of</strong> the patterns onto the photoresist film via photomasks. The resist’s
solubility alters on the exposed areas.
(d) Dissolution <strong>of</strong> the exposed areas <strong>of</strong> the photoresist with ammonium
hydroxide.
(e) Chemical etching that removes the metal that extends under the exposed
photoresist. Dissolution <strong>of</strong> the unexposed areas <strong>of</strong> the photoresist in order to
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eveal the metal patterns, which extend underneath. It follows plasma
treatment that removes the organic remains and cleans up the substrate.
(Alternative to chemical etching is the lift-<strong>of</strong>f process, where metal sputters
the top <strong>of</strong> a photoresist film and covers only the uncoated areas <strong>of</strong> the
substrate. It follows detachment <strong>of</strong> the unexposed areas from the substrate.)
(f) Spin-coat SU8 photoresist, at typical speed <strong>of</strong> 1500 rpm for 30 s, and
hardening at 90 °C for 2 min. The SU8 layer may develop up to thicknesses
<strong>of</strong> some hundred micrometers. This SU8 layer is used as a material for
producing microchannels.
(g) Transferral <strong>of</strong> patterns onto the SU8 layer by ultraviolet exposure <strong>of</strong> the SU8
via chromium photomask. The SU8 resist is then developed with methoxypropyl-acetate
that removes the unexposed SU8 areas and reveals the
microchannels. (Alternative to SU8, microchannels might be directly etched
on glass substrate by means <strong>of</strong> hydr<strong>of</strong>luoric acid etcher.)
(h) Dishing <strong>of</strong> the substrate in order to separate the individual chips and drill <strong>of</strong>
inlets.
(i) Enclosure <strong>of</strong> the chip by aligning and crosslinking two opposite plates at the
temperature 150 °C for 30 min.
S<strong>of</strong>t lithography is an alternative fabrication technique, based on direct
printing or molding <strong>of</strong> polymers [ 126,127 ]. In s<strong>of</strong>t lithography, instead <strong>of</strong> stiff
photomasks, elastomers are used as stamps, molds, or masks, in order to replicate
patterns (Fig. 4). In s<strong>of</strong>t lithography the silicone elastomer PDMS (polydimethyl-
Fig. 3. Photolithographic development <strong>of</strong> microelectrodes and microchannels on a wafer substrate:
(a) metallization <strong>of</strong> the substrate by sputtering a metal film <strong>of</strong> Au, Pt, or ITO; (b) spin coating <strong>of</strong>
photosensitive resist film onto the metal film; (c) exposure <strong>of</strong> the photosensitive film via a
photomask that results in the transfer <strong>of</strong> the desired electrode patterns onto the photosensitive film;
(d) after photo-development, chemical etching removes the bare metallized areas, which results in
the formation <strong>of</strong> the electrodes; (e) spin coating <strong>of</strong> an SU8 photoresist layer; (f) exposure <strong>of</strong> the
SU8 photoresist film via photomasks and development; (g) chemical etching and removal <strong>of</strong> the
unwanted SU8 resist. The revealed half microchannel has sidewalls made <strong>of</strong> the SU8 material.
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Fig. 4. Sequential steps <strong>of</strong> the s<strong>of</strong>t fabrication process, which can produce features <strong>of</strong> a few
hundreds <strong>of</strong> microns: (a) a hard master substrate made <strong>of</strong> metal, glass or plastic, casts on a mold
material which can be polymer, thermoplastic, PDMS or PMMA; (b) upon heating and
pressurization the mold deforms to the shape <strong>of</strong> the master; (c) lifting <strong>of</strong>f the master reveals the
microchannels.
siloxane) is widely preferred because <strong>of</strong> its easiness to cast, its biocompatibility,
hydrophobicity, and sealing properties. S<strong>of</strong>t lithography is recommended only for
rapid prototyping in research. Because <strong>of</strong> the s<strong>of</strong>t materials used in s<strong>of</strong>t lithography,
distortions <strong>of</strong> stamps and molds are problems that prevent s<strong>of</strong>t lithography
from becoming a viable manufacturing technique. For example, the widespread
PDMS swells when it comes in contact with oleic solvents.
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5. MATERIALS AND METHODS
5.1. Fabrication materials
Lab-on-chip fabrication techniques are analogous to those <strong>of</strong> microelectronics,
since closely related micr<strong>of</strong>abrication and integration methodologies
are shared by both. The very first fluidic devices were fabricated on silicon (Si)
substrates by means <strong>of</strong> silicon integration technology and afterwards a more
advantageous material, glass (SiO2), was introduced that demonstrates robustness
and better electrical insulation [ 128–132 ]. Modern <strong>lab</strong>-on-chip devices are hybrids
that combine glass, silicon and various polymers like acrylic, polyester, polycarbonate,
resists, thermoplastics or molds like the polydimethylsiloxane
(PDMS). Precision, miniaturization, cost effectiveness, large-scale production
and ability <strong>of</strong> incorporating electronics make these materials very attractive.
They are proven very reliable since they demonstrate strong crystalline structures
and can survive long-term use.

Silicon, glass or polymers are suitable for making the micr<strong>of</strong>luidic components
<strong>of</strong> the chips; metals like gold, platinum or titanium are used for the conductive
parts; silicon dioxide, silicon nitride and titanium nitride are for insulation
and passivation; electroactive polymers or other elastomers are for the
moving components. The standard procedure <strong>of</strong> producing fluidic chips on
silicon or glass substrates involves:
(a) deposition <strong>of</strong> metallic and dielectric layers on silicon or glass wafer;
(b) patterning these layers by means <strong>of</strong> photolithography in order to transfer
onto them the outlines <strong>of</strong> the microchannels, conductive or insulated areas,
or other features;
(c) chemical etching that removes the unwanted material and reveals the desired
structures.
Specifically for capillary electrophoresis devices, the use <strong>of</strong> silicon substrates
is limited by their semiconducting property; due to the high electric fields
required for performing capillary electrophoresis, electric shorting very likely
occurs. For this reason, glass substrates are mostly preferred for making capillary
electrophoresis devices.
Polymers, like polycarbonates, have varied interfacial and structural characteristics.
Polymers have disadvantageous optical properties in comparison to
glass, like lower light transmittance and higher self-fluorescence emission, but
better thermal conductance. Micr<strong>of</strong>luidic channels can be made <strong>of</strong> polymers by
means <strong>of</strong> molding or micromachining [ 133,134 ]. Particularly fluoropolymers, such
as polytetrafluoroethylene (PTFE (C2F4)n) are handy materials for structuring
micr<strong>of</strong>luidic channels as they are s<strong>of</strong>t and can be easily milled, and are also
hydrophobic, which highly eases liquids to flow within the microchannels. For
rapid prototyping, casting <strong>of</strong> polydimethylsiloxane (PDMS (C2H6OSi)n), by
means <strong>of</strong> silicon or smoothed metallic masters, can produce PDMS micr<strong>of</strong>luidic
channels [ 135 ]. PDMS provides flexibility, hydrophobicity and very tight sealing
under application <strong>of</strong> uniform mechanical pressure. PDMS is capable <strong>of</strong> sealing
various smooth surfaces, including glass, silicon, silicon nitride, polyethylene,
glassy carbon, oxidized polystyrene, fluorocarbons and metals. Since s<strong>of</strong>t lithography
is usable for fast prototyping, PDMS is preferred in s<strong>of</strong>t lithography as an
easy-to-cast material for outlining microchannels on smoothed substrates.
Compared to glass, PDMS has lower thermal conductivity, much higher hydrophobicity,
and both these properties qualify PDMS as a suitable material for
making micr<strong>of</strong>luidic channels. On the other hand, PDMS is swollen by many
organic solvents like oils, but remains unaffected by water, nitromethane, ethylene
glycol, acetonitrile, perfluorotributylamine, perfluorodecalin and propylene
carbonate. Furthermore, PDMS is known to absorb small lipophilic molecules [ 136 ].
The compatibility <strong>of</strong> PDMS against organic solvents can be enhanced by means
<strong>of</strong> coating the PDMS surface with sodium silicate. Yet, due to the pliability <strong>of</strong>
PDMS, electrodes cannot be patterned on it [ 1 ].
Metal films like gold (Au), platinum (Pt) and titanium (Ti), or metalloid films
like Indium Tin Oxide (ITO), are appropriate for making microelectrodes for <strong>lab</strong>-
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on-chip devices. Gold and platinum are highly biocompatible, chemically inert
and thus are suitable metals for contacting biochemical solutions. Thin gold,
platinum, titanium, or ITO films may be sputtered, or deposited by means <strong>of</strong>
evaporation, on wafer substrates. The electrode patterns subsequently can be
revealed by chemical etching or lift-<strong>of</strong>f process, which allows resolution <strong>of</strong> few
micrometers. The chemical etching method is analysed below in Section 5.4.
Alternative to chemicals, it is possible to reveal electrodes by means <strong>of</strong> precise
milling <strong>of</strong> the metallized substrate at resolution <strong>of</strong> few tens to hundreds <strong>of</strong>
micrometers. The electrodes <strong>of</strong> micr<strong>of</strong>luidic devices are usually coated with a
dielectric film because the dielectric coating prevents electrolysis <strong>of</strong> the contacting
fluids. But in the case <strong>of</strong> very low voltage applications, such as bioimpedance,
the electrodes can remain bare. Other usages <strong>of</strong> metal films in <strong>lab</strong>-onchips
technology entail production <strong>of</strong> masks, overlayers, or adhesion layers. Yet,
micr<strong>of</strong>luidic heaters or heat elements <strong>of</strong> Pt / Ti can be structured with 200 nm Pt,
electrodeposited onto 20 nm Ti film. Pt thin films on glass substrate are capable
<strong>of</strong> withstanding thermal bonding and thus Pt is preferred for wiring contacts [ 1 ].
For controlling liquid flows, movable micro-apparatuses might be integrated
within <strong>lab</strong>-on-chip devices. The electroactive polymers are an important class <strong>of</strong>
elastomers for fabricating microvalves or micropumps, as they can be precisely
controlled electrically.
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5.2. Coating materials
The metallization <strong>of</strong> silicon, glass or polymer substrates is a process which
precedes the patterning <strong>of</strong> the electrodes. Metal films can be sputtered or
chemically deposited on silicon, glass or polymer substrates. Gold and platinum
are two metals widely adopted by the chipmakers in the production <strong>of</strong> microelectrodes,
due to two significant properties:
(a) excellent conductivity, which allows transmission <strong>of</strong> electrical signals without
distortions;
(b) biocompatibility, which allows direct contact with biochemical solutions
without adhesions and adsorptions.
The density <strong>of</strong> a metal film that is developed by means <strong>of</strong> chemical deposition
is a bit lower than that <strong>of</strong> bulk metals. The deposited metal film might contain
defects such as pores and inclusions <strong>of</strong> foreign matter <strong>of</strong> 2–30 nm in diameter.
Further, its mechanical strength depends on its chemical composition, deposition
rate and temperature and pressure conditions. For instance, mechanical strengths
<strong>of</strong> 40–50 kg/mm 2 might be obtained at 50–70 °C [ 137 ]. The electrical conductivity
<strong>of</strong> chemically deposited metal films is usually lower than that <strong>of</strong> pure metals. The
optical properties, however, are less varied and do not differ much <strong>of</strong> those <strong>of</strong>
pure metals. The optical characteristics <strong>of</strong> a fabrication material should be considered
in those <strong>lab</strong>-on-chip devices that work with optical sensing apparatuses.
In the case when a micr<strong>of</strong>luidic device integrates optical sensors, the micr<strong>of</strong>luidic
channels <strong>of</strong> this device should be made transparent in order not to disrupt the

light beam. Transparent electrodes like indium-tin-oxide might be required in the
case when electrodes and optical mechanisms are combined on the same micr<strong>of</strong>luidic
channel.
Yet, it is possible to develop gold electrode patterns <strong>of</strong> micrometer thickness
on polymer substrates by means <strong>of</strong> aerosol spraying metallization, where gold
and amines, combined together with hydrazine as reducer, produce gold films <strong>of</strong>
thickness <strong>of</strong> 400 nm/min [ 137 ]. Similarly, platinum can be deposited adjustably at
rates <strong>of</strong> 500–2000 nm/h with borohydride or hydrazine as reducing agents.
Oxide films <strong>of</strong> nanometer thickness are usable as electrode insulators, because
they prevent hydrolysis <strong>of</strong> fluids. It is possible to accurately grow oxide layers on
electrodes by means <strong>of</strong> deposition, sputtering, or anodic or thermal oxidation.
The oxide films further protect the electrodes from chemical aggressions,
especially those electrodes that come to direct contact with electrolytes,
biological solutions or acids. An oxide protective film prevents electrolysis
because <strong>of</strong> high resist against leakage currents, produced by high-voltage electrokinetic
phenomena in fluids (e.g. electrophoresis, dielectrophoresis, electrowetting).
However, for low voltage impedimetric applications, mV or below, it is
feasible to directly contact the liquid sample with bare electrodes. Thermally
grown oxide films, like silicon dioxide (SiO2) and the self-healing tantalum
pentoxide (Ta2O5), are appropriate options for robust electric insulation due to
their high breakdown resistance [ 51,138,139 ]. Thermally grown SiO2 layers may
form thicknesses <strong>of</strong> some nanometers. SiO2 and Ta2O5 can, alternatively, be
deposited by plasma, or be sputtered on metal electrodes. However, compared to
the deposited ones, the anodically or thermally grown oxide nan<strong>of</strong>ilms are more
effectual in terms <strong>of</strong> adhesion and electric insulation, due to their layer’s
continuity with the crystalline structure <strong>of</strong> the metal electrode and their low
pinhole density.
Ceramic coating films, like silicon nitride (Si3N4), can be deposited by means
<strong>of</strong> chemical vapor deposition, or plasma-enhanced chemical vapor deposition.
Si3N4 films are used as electric insulators and are superior to SiO2, but weaker
than Ta2O5. Strontium titanate (SrTiO3) has very large permittivity and thus it is
suitable for high voltage electric insulation.
Transparent electrodes, like Indium Tin Oxide, are favoured in <strong>lab</strong>-on-chip
manufacturing, because their transparency allows microscopy. Indium Tin Oxide
films are mostly deposited on substrates by means <strong>of</strong> electron beam evaporation,
physical vapor deposition or sputtering.
The hydrophobization <strong>of</strong> micr<strong>of</strong>luidic channels enables repulsion <strong>of</strong> aqueous
fluids and improves their flow, especially the droplet-based segmented flows.
Hydrophobic films are very suitable as coatings for microchannels. Different
hydrophobization methods have been introduced, ranging from spontaneous
covalent bonding <strong>of</strong> hydrophobic self-assembles <strong>of</strong> organometallic octadecyltriclorosilanes,
a process called silanization, to chemical and plasma deposition <strong>of</strong>
fluoropolymers [ 140–146 ]. Alternatively, instead <strong>of</strong> hydrophobizing a microchannel,
it is possible by introducing hydrophobic surfactant into aqueous droplets to
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encapsulate them into surfactant membranes. Supreme surfactants are the fluorosurfactants
(CnF2n+1)n [ 147 ].
Prior to coating processes, some chemical treatment in the interior <strong>of</strong> a
microchannel can enhance the adhesion <strong>of</strong> a coating material onto the microchannel.
For example, a glass microchannel can be cleaned with ethanol, acetone,
or a mixture <strong>of</strong> strong acids. Likewise, in polymeric microchannels the adhesion
<strong>of</strong> a film develops faster on prepared and cleaned surfaces and this results in
lasting coatings. Some molded thermoplastics, processed in high melt temperature,
followed by rapid cooling, are easier to coat because their surface activity
increases adhesion. Plasma treatment also cleans organic matter from the interior
<strong>of</strong> a micr<strong>of</strong>luidic chip. Plasma cleaning can either be performed before assembling
the chip or afterwards. In the latter case capillary plasma can be produced
within the fluidic channels by following the steps:
(a) seal the microchannel inlets with needle electrodes,
(b) pump out the air from the microchannel in order to generate vacuum,
(c) apply high voltage pulses, <strong>of</strong> few thousands <strong>of</strong> volts, to initiate electric
sparks, which rapidly evolve into plasma arc, which can become visible
through the transparent microchannel <strong>of</strong> the chip. The plasma can be
produced with reaction gases such as neon, helium and argon.
Treatment using strong acids can functionalize the inner surface <strong>of</strong> a micr<strong>of</strong>luidic
channel. For example, introducing nitric acid into a glass microchannel it
can functionalize the hydroxyl groups <strong>of</strong> the glass to strengthen the bond between
octadecylitroclorosilanes and the glass and consequently produce a uniform
hydrophobic thin layer inside the microchannel.
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5.3. Deposition methods
Chemical vapour deposition is a process that produces thin metal, ceramic, or
compound films, through thermal oxidation in a gas chamber at an elevated
temperature. Within the chamber the substrate interacts, at temperatures between
800–2000 °C and pressures between millitorrs to torrs, with volatile precursors
that react and decompose on the substrate a film <strong>of</strong> a metal (e.g. Al, Ta, Ti, Pt),
ceramic (e.g. Si3N4, B2O3, BN) or compound. The gas mixture typically consists
<strong>of</strong> reducing gas, like hydrogen (H2), inert gases like nitrogen (N2) or argon (Ar),
and reactive gases such as metal halides and hydrocarbons. A typical chemical
reaction sequence includes pyrolysis, reduction, oxidation, hydrolysis and
coreduction. For example, silicon nitride (Si3N4) can be deposited by means <strong>of</strong>
reaction <strong>of</strong> dichlorosilane gas (SiCl2H2) with ammonia gas (NH4) at temperatures
between 700–800 °C [ 72 ]. The volatile byproducts can be blown away from the
reaction chamber and neutralized before exposure to the environment. The
chemical vapour deposition can also be plasma enhanced, a method that functionalizes
surfaces and is effective in depositing hydrophobic films on wafers.
For example, a fluorocarbon hydrophobic film can be developed on an interface
by means <strong>of</strong> plasma-enhanced chemical vapour deposition <strong>of</strong> CHF3. The plasma-

enhanced technique can also be used in the case <strong>of</strong> depositing insulating films
such as silicon nitride (Si3N4) or plasma coated silicon dioxide (SiO2). A similar
method, capillary plasma, produces gas plasma within a microchannel, by sealing
its inlets with electrode needles, pumping out the air from an outlet, inserting the
reaction gases and applying high voltage pulses across the needles. In this way a
plasma arc is produced that can be clearly visible if the channel is transparent.
Capillary plasma deposition <strong>of</strong> platinum bisacetylacetonate precursor is performed
for deposition <strong>of</strong> platinum inside channels. The closely related technique
<strong>of</strong> plasma electrolytic oxidation, or microarc oxidation, can electrochemically
produce on top <strong>of</strong> an electrode an oxide coating <strong>of</strong> micrometer thickness, which
provides electric insulation with excellent adhesion.
Adsorption is a chemical deposition method that exploits hydrophilic head
groups <strong>of</strong> self-assembled monolayers <strong>of</strong>, for example, hydrophobic trichlorosilanes
(Cl3SiCnH2n+1), or thiols (HSCnH2n+1), which <strong>of</strong>fer spontaneous binding on
glass or metal surfaces [ 148 ]. A glass substrate can be functionalized by acidic
treatment with a mixture <strong>of</strong> sulfuric acid (H2SO4) and hydrogen peroxide (H2O2),
which is well known for producing silanol groups on glass. Silanes bind
covalently to the hydroxyls <strong>of</strong> silanols, with glass being the most obvious surface
with silanol groups.
Physical vapour deposition, such as sputtering or evaporation, is used to
deposit thin films, layer by layer, onto substrates. This method employs mechanical
or thermodynamic means for producing thin films and requires low-pressure
vaporized environment to function.
Ion beam enhanced deposition influences the energy and charge states <strong>of</strong> the
gas in the vapour phase and allows control over the energy state and crystallographic
and stoichiometric form <strong>of</strong> the deposited films.
5.4. Etching procedures
In <strong>lab</strong>-on-chip fabrication technology, patterning is the transfer <strong>of</strong> outlines <strong>of</strong>
features (which define microchannels, microelectrodes, or other components) on
the top <strong>of</strong> a substrate by means <strong>of</strong> ultraviolet illumination via a photomask. The
photomask consists <strong>of</strong> a chromium layer where only part <strong>of</strong> it is transparent to the
light. Photolithography is the method for producing structures by exposing
photosensitive resists to ultraviolet light via successive photomasks and then
removing the exposed areas <strong>of</strong> the photoresist in a development process. The
photosensitive resist is a polymer, usually epoxy, that is sensitive to ultraviolet
light. When the ultraviolet exposes specific areas, the photoresist polymerizes
and resists to etchers. This replicates the patterns onto the photoresist film. The
exposed areas <strong>of</strong> the resist film are being attacked by the developer and
consequently removed. The unexposed areas remain resistant to the action <strong>of</strong> the
etchant. The structure can be revealed after etching the exposed areas. Every
wafer substrate may undergo many sequential etching cycles before completes.
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The resolution <strong>of</strong> the features that can be structured with photolithography can be
some micrometers. The major steps <strong>of</strong> the etching process are shown in Fig. 5.
We distinguish between two etching processes, wet etching and dry etching.
Wet chemical etching is widely used for producing microelectrodes and
micr<strong>of</strong>luidic channels on substrates. The wet etching requires acids, bases or
mixtures to dissolve metals, silicon or glass, by immersing the substrate into the
etching solution. For instance, gold can be etched using iodine solution. The
etching can be isotropic or anisotropic. As an example, silicon can be
etched isotropically with HF-HNO3, which produces rectangular curved grooves
by means <strong>of</strong> thermally grown SiO2 mask. KOH can etch Si or Si
anisotropically, with the etching rate depending on the crystallographic orientation,
and produces grooves with 55° or 90° walls, respectively. It is important to
mention, however, that with anisotropic etching the exact defined shape is
difficult to be obtained because <strong>of</strong> the directional dependence <strong>of</strong> the etching rate,
and so this method is limited to small dimensions only. Glass can be etched
isotropically using buffered hydr<strong>of</strong>luoric acid (HF) in HCl or ultrasonic bath that
improves smoothness <strong>of</strong> the etched surface. Removal rates <strong>of</strong> glass are typically
between 0.1–1.0 µm/min. The etchant should be selected carefully in order to
have the etching rate <strong>of</strong> the masks considerably lower than that <strong>of</strong> the removable
material. The final step in a wet etching process is to remove the resist by
washing it away with an organic solvent.
Dry etching involves reactive ion etching with plasma, where the substrate is
placed into a plasma chamber where a gas mixture is introduced and is ionized.
The ionized gas mixture reacts with the surface <strong>of</strong> the substrate to be etched. As
the ionized gas is highly energized, it removes the matter from the substrate.
Xenon difluoride (XeF2) is a dry vapour phase isotropic etcher for silicon.
Fig. 5. Sequential steps <strong>of</strong> a chemical etching process, which can produce microchannels with
width <strong>of</strong> some tens to hundreds <strong>of</strong> microns: (a) exposure <strong>of</strong> a photoresist-coated substrate to
ultraviolet light; (b) dissolution <strong>of</strong> the exposed areas <strong>of</strong> the photoresist with chemical developer;
(c) chemical etching <strong>of</strong> the bare areas <strong>of</strong> the substrate; (d) revelation <strong>of</strong> the microchannel after total
removal <strong>of</strong> the photoresist from the substrate.
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Its etching selectivity to silicon is very high and does not affect masks made <strong>of</strong>
photoresists, silicon dioxide, silicon nitride or metals. For example, the
selectivity to silicon nitride is better than 100 : 1 and reported to be as high as
(5000–10 000) : 1 for silicon dioxide [ 149 ]. SF6 in high-density plasma provides
anisotropic high-aspect-ratio etching for silicon. Typical dry etching rates for
silicon are around 2 µm/min.
A suitable photoresist for structuring fluidic microchannels on wafer
substrates is the biocompatible and chemically inert epoxy SU8, which develops
vertical sidewalls <strong>of</strong> micrometer height on glass or silicon wafers [ 150,151 ]. The
SU8 photoresist can be spun onto the wafer substrate using spin-coater at a speed
that achieves specific thickness, under certain viscosity value and specific
hydrophobic surface. For example, with usual viscosity and hydrophobicity, to
obtain 100 µm thick layer <strong>of</strong> SU8, the wafer substrate should be spun at 500 rpm
for 5 s, to spread out the photoresist, then at 3000 rpm for 30 s, to achieve the
final thickness. The substrate should then be heated for 10 min at 65 °C, and for
30 min at 95 °C. The two-step bake is required in order to reduce internal stresses
owing to thermal effects. The photoresist is then exposed in ultraviolet via a
photomask. Solidifying one SU8 formation against another by crosslinking the
opposing SU8 layers and baking at 65–95 °C, it is viable to produce microchannels.
The low surface roughness <strong>of</strong> SU8 enables the use <strong>of</strong> optical analytical
techniques such as fluorescence or optical spectrometry. The possibility <strong>of</strong>
integrating microelectrodes on SU8, the possibility <strong>of</strong> hydrophobizing the SU8
and <strong>of</strong> bonding it at low temperature, makes SU8 a particularly suitable resist for
assembling <strong>lab</strong>-on-chip devices. Furthermore, SU8 is suitable <strong>of</strong> serving as mask
on the top <strong>of</strong> the silicon wafer in wet etching processes [ 1 ].
6. BONDING
After patterning all features on substrates (microchannels, elements, inlets,
etc), the base plate and the cover plate must be bonded in order to seal the chip. It
is possible to bond silicon, glass, or rigid polymer plates, by three different
means.
Anodic bonding can fuse silicon or glass plates. The two opposing plates must
come in contact with each other, in heat, with a high voltage applied across a
conductive layer (200 nm silicon nitride (Si3N4) or 120 nm Ni/Cr) developed
intermediately, that causes diffusion <strong>of</strong> ions, which eventually fuses the two
plates electrostatically. Anodic bonding conditions vary between 200–1500 V, at
200–450 °C.
Thermal bonding can fuse glass or polymer plates. Thermal bonding is
performed on coated with methylsilses-quioxane, or polysiloxane, surfaces at
temperatures 150–210 °C. Annealing for some hours in low vacuum produces
bonds that withstand hundreds <strong>of</strong> N/cm 2 . Rapid glass bonding requires hot
pressure at 570 °C for 10 min under 4.7 N/mm 2 .
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Photopolymer adhesives can bond any type <strong>of</strong> rigid substrates. This method is
more tolerant for uneven surfaces since it provides a compressible cushioning
layer that seals the chip. The adhesive layer can be benzocyclobutene (C8H8) or
UV-curable resins <strong>of</strong> micrometer thickness. It is possible to heat and separate the
plates apart and rebond.
To sandwich elastomers, such as PDMS, between plates, mechanical pressure
must be applied uniformly. Uniform pressure is usually obtained by squeezing
with a metal holder the plates against the elastomer by means <strong>of</strong> screws. The
advantage is the possibility <strong>of</strong> reopening the chip.
128
7. FLUIDIC AND ELECTRIC CONNECTIONS
Customized chip holders can support fluidic tube connections and wired
electrical connections. Flanged plastic fittings having standard threads can be
used for connecting the tube. This connector type uses a metal O-ring, which
presses the flanged tip <strong>of</strong> the tube against the inlet <strong>of</strong> the chip and seals the inlet
tightly after screwing into the respective threads <strong>of</strong> the chip holder. Another
connection method uses injection-molded connectors with internal threads that
can withstand pressure <strong>of</strong> 1 MPa. Furthermore, adhesive or solder-based chip-totube
connections can tolerate elevated pressures and solvents.
8. BIOCOMPATIBILITY AND DEFECTS
Biocompatibility concerns the safety <strong>of</strong> the material and its performance to
meet some standards in order to avoid harming the biological substances that
come in contact with the material <strong>of</strong> a device. The biocompatibility <strong>of</strong> <strong>lab</strong>-onchip
devices came forth with the advent <strong>of</strong> implantable biochips. Biocompatibility
attempts to avoid materials that cause cytotoxicity, sentitization,
irritation, genotoxicity, carcinogenicity or other abnormalities [ 88 ]. The biocompatibility
<strong>of</strong> a device concerns only the materials that come into contact with
biological substances.
There are numbers <strong>of</strong> possible defects, which should be considered when
designing <strong>biomedical</strong> <strong>lab</strong>-on-chip devices. Since most <strong>of</strong> the micr<strong>of</strong>luidic
channels are made hydrophobic, most enzymes, proteins and cells adsorb on
hydrophobic areas, an effect called bi<strong>of</strong>ouling. Bi<strong>of</strong>ouling disorders the micr<strong>of</strong>luidic
chips, because it alters their microchannels into hydrophilic, which affects
the laminar flow. Bi<strong>of</strong>ouling affects sensing elements, such as microelectrodes,
which consequently affects the accuracy <strong>of</strong> the measurements. Silicon, silicon
nitride, silicon dioxide, gold, platinum, titanium, SU8 and photoresists are
biocompatible and all <strong>of</strong> them have reduced bi<strong>of</strong>ouling and cytoxicity [ 152–154 ].
Particularly, silicon nitride is used for long-term implantations.

The proteins bovine-serum-albumin (BSA) and casein (phosphoprotein)
reduce the adhesion <strong>of</strong> enzymes and proteins on the interfaces. Important
advantage <strong>of</strong> bovine-serum-albumin and casein is their biocompatibility with
most <strong>of</strong> the proteins.
To prevent adhesion <strong>of</strong> cells in micr<strong>of</strong>luidic channels, surfactants like
poloxamer copolymers can be added inside the channels [ 155 ]. However,
poloxamers have shown to incorporate into the cellular membranes, particularly
<strong>of</strong> the cancerous cells, affecting so the microviscosity <strong>of</strong> the cell membrane.
9. FUTURE DIRECTIONS
New <strong>lab</strong>-on-chip devices must emerge that demonstrate a positive impact on
clinical diagnostics. The trend is to improve sterility, multiple analysis and
parallel processing with increased efficiency and speed. Future <strong>lab</strong>-on-chip
devices will consume ultralow power, will be smaller and lighter with more
emphasis on the user comfort. Improvements in portability should emphasize
easy sample introduction and fluidic connectivity. Communication technologies
like wireless networking, information management and advanced user interface,
visualization and navigation technologies through adapted screens will be
embedded. Standardization will condition device reliability, interoperability,
biocompatibility, electromagnetic compatibility, interconnectivity, readouts,
weight and size [ 156 ].
The functionality <strong>of</strong> future <strong>lab</strong>-on-chip devices is foreseen to improve clinical
tests and will be shifted from general-purpose <strong>lab</strong>oratory instruments to strictly
personalized devices. To date the <strong>lab</strong>-on-chip devices are determined by applications
in bioanalysis. Additionally to mixing, measuring, sorting or separation,
micr<strong>of</strong>luidic <strong>lab</strong>-on-chips are foreseen to facilitate on-chip supramolecular
chemistry, <strong>biomedical</strong> implants, biomimetics, and hybrid systems incorporated
with blood vessels [ 157 ]. Future applications will certainly include nanobiotechnologies.
Envisions consider nanoscaled channels, where surface effects,
rather than volume, dominate liquid behaviour. Chemical synthesis <strong>of</strong> macromolecules
will be actualized molecule-by-molecule, ensuing fully control<strong>lab</strong>le
and accurate synthesis <strong>of</strong> bioproducts, independent <strong>of</strong> the constraints <strong>of</strong> statistics
and diffusions parameters that rule present chemistry in vessels.
Experts claim that the complexity <strong>of</strong> <strong>lab</strong>-on-chip devices is increasing at rates
comparable to Moore’s law. Most <strong>of</strong> the present <strong>lab</strong>-on-chip devices operate with
external computing units. The future trend is to develop fully standalone <strong>lab</strong>-onchip
devices with embedded computing and calibrating capabilities. Further
development <strong>of</strong> micr<strong>of</strong>luidic biocomputers will take place that will use biological
macromolecules, such as nucleic acids or proteins, to perform computations
including storing, retrieving, and biological data processing through application
<strong>of</strong> biochemical principles [ 158 ]. DNA computation employs massive parallelism
inherent in the small scale <strong>of</strong> molecules to speed up decisions. The hybridization
129

<strong>of</strong> nucleic acids can be either exploited to encode strands <strong>of</strong> nucleotides to clone
nucleic acids, or to self-assembly oligonucleotides. Emphasis is given to improving
methods for hybridization by surpassing various physicochemical constraints
and use DNA to compute in environments where it is uniquely capable <strong>of</strong>
operating, such as smart drug delivery to individual cells [ 159,160 ]. Further variations
in biocomputing use proteins instead <strong>of</strong> nucleic acids [ 161 ], or DNA hairpin
formation [ 162 ], self-assemblies and cellular computation, in which cells are
considered computational elements.
Networking individual <strong>lab</strong>-on-chip devices is foreseen to boost <strong>lab</strong>-on-chip
functionalities, as information can be exchanged between individual devices via
computer servers in hospitals. Experts speak about interconnecting <strong>lab</strong>-on-chips
to the patients’ databases for online updating their medical records and to perform
classification <strong>of</strong> the diseases. Networking biochips can benefit telemedicine,
where <strong>lab</strong>-on-chip testers will be capable <strong>of</strong> transferring test results to
doctors entirely remotely. The upcoming generations <strong>of</strong> <strong>lab</strong>-on-chip devices will
be cabled or wirelessly interconnected in network-centric data links, being
synchronized as terminal testers for the same or different patients, located at a
distance. In rural and urban areas <strong>lab</strong>-on-chip technology can contribute to
ubiquitous monitoring the diseases, pathogens, or toxic substances in the environment.
For example, miniaturized biochemical marine sensors, submerged in seawaters,
can sample contaminants in the oceans. The functionality <strong>of</strong> such
environmental biochips is based on various principles including monitoring
oxygen levels, cell analysis <strong>of</strong> phytoplankton, observation <strong>of</strong> methane, nitrate and
phosphates in seawaters [ 163–166 ]. Moreover, autonomous integrated biochips may
be distributed in public areas, in buildings, airports, subways, parks, stadiums,
schools, and stores, to collect biochemical information and detect the onset <strong>of</strong>
major infectious pathogens or record signs <strong>of</strong> pollutions, either on air, sea or
land.
The impact <strong>of</strong> <strong>lab</strong>-on-chip devices in everyday life is expected to be
analogous and as revolutionary as integrated circuits and microelectronics. Labon-chip
devices will irrevocably influence medicine, chemistry, biology, biotechnology
and bioelectronics.
130
10. CONCLUSIONS
The most appropriate materials, fabrication techniques and assembling
methods for manufacturing <strong>lab</strong>-on-chip devices were reviewed in this article.
Lab-on-chip devices must be made <strong>of</strong> materials that are biocompatible,
chemically inert, reliable and reusable for lifetime. Presently, gold or platinum
are most suitable for structuring conductive elements. Fluoropolymers and
silanes are the most suitable for hydrophobizing fluid-solid interfaces. Oxidegrown
films or plasma-deposited films, particularly the thermally or anodically
grown SiO2 and Ta2O5, are the most efficient for insulating the conductive
elements <strong>of</strong> the <strong>lab</strong>-on-chip devices.

Highly reliable <strong>lab</strong>-on-chip devices are still a long way ahead. The complexity
and technological problems to be solved are enormous. Major obstacle is the
technological limitation <strong>of</strong> the present mic<strong>of</strong>abrication materials. Until new revolutionary
materials are introduced, any tremendous development in comparison to
the present state should not be expected. The to date development <strong>of</strong> <strong>lab</strong>-on-chip
devices is analogous to the state <strong>of</strong> the electronics before the discovery <strong>of</strong>
semiconductors. However, once new synthetic materials become avai<strong>lab</strong>le,
especially new nanomaterials, an enormous boost, analogous to this <strong>of</strong> microelectronics
in the semiconductors era, is anticipated. It means that <strong>lab</strong>-on-chip
designers are awaiting innovations from material science and chemistry to solve
reliability, biocompatibility and integration problems. Furthermore, <strong>lab</strong>-on-chip
technology acquires improvements in the device production methods, and is
forecasted to acquire specific technologies from physical sciences and other
disciplines <strong>of</strong> engineering, as well as from biology [ 167 ].
Conditio sine qua non <strong>of</strong> future <strong>lab</strong>-on-chip device design are the following
very specific requirements and standards:
(a) biocompatibility, robustness, sensitivity, and reliability;
(b) low power consumption, high degree <strong>of</strong> automation and integration, and
high sample throughput;
(c) coverage <strong>of</strong> a broad range <strong>of</strong> biological samples and measurement parameters.
ACKNOWLEDGEMENTS
Acknowledgements are due to Dr Uwe Pliquett and Dr Brian Cahill for
technical discussions. Financial support comes from a European Commission
Marie Curie ERG (project PERG06-GA-2009-256305) and an Estonian Science
Foundation Mobilitas Grant (project MOJD05).
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Biomeditsiiniliste kiip<strong>lab</strong>orite valmistamine
Athanasios T. Giannitsis
Kiip<strong>lab</strong>orid on miniatuursete analüütiliste seadmete klassi kuuluvad seadmed,
mis ühendavad endas voolise, elektroonika ja sensoorika ning on ette nähtud biokeemiliste
vedelike analüüsimiseks. Täiendavate abivahenditena kergendavad
kiip<strong>lab</strong>orid vedelike transporti, sorteerimist, segustamist või lahutamist. Nende
miniatuursete seadmete tähtsus ilmneb <strong>lab</strong>oratoorsete protseduuride automatiseerimises,
mis vähendab tunduvalt biomeditsiiniliste katsete ja <strong>lab</strong>oratoorsete toimingute
aega. Antud ülevaateartiklis on kirjeldatud arvukaid kiip<strong>lab</strong>orite valmistamise
meetodeid ja protseduure, samuti on vaadeldud võimalikke tulevikuarenguid.
139